Journal of Clinical InvestigationVol. 41, No. 10, 1962

RELATIONSHIP BETWEENURINARY HYDROXYPROLINEANDGROWTH*

By H. E. JASIN,t C. W. FINK, W. WISE, AND M. ZIFF

(From the Departments of Internal Medicine [Rheumatic Diseases Unit] and Pediatrics, Uni-versity of Texas Southwestern Medical School, and the Arthritis Clinical

Study Center, Parkland Memorial Hospital, Dallas, Tex.)

(Submitted for publication May 18, 1962; accepted July 3, 1962)

Beginning with the report of Ziff, Kibrick,Dresner, and Gribetz (2), evidence accumulatedin recent years has indicated that urinary hydroxy-proline excretion reflects collagen metabolism,since collagen is the only protein in the body thatcontains hydroxyproline in significant amounts(3). Ziff and co-workers, observing that theexcretion of this amino acid in peptide form wasgreater in children than in adults, suggested thatthe level of urinary hydroxyproline might be re-lated to the size of the metabolically active, solu-ble collagen pool available for breakdown. Theresults of a number of investigations (4-7) havesupported this suggestion.

In the present paper, further investigation ofthe relationship between growth and hydroxypro-line excretion has been carried out in man. Uri-nary hydroxyproline has been determined in pa-tients with normal, arrested, and acceleratedgrowth, and in cases in which arrested growthhas been stimulated by hormonal therapy. Thedata obtained indicate that the level of excretionof hydroxyproline shows a good correlation withrate of growth under all circumstances studiedand, therefore, offers a useful and readily meas-ured index of this phenomenon.

METHODSANDMATERIALS

Normal controls. Urines from normal children andadults were collected at home. In the 0- to 1-year group,the urine was collected in absorbent diapers with aspecial tissue paper sheet to isolate the feces. Diaperswere changed six times in 24 hours. Upon removal,they were immersed in water saturated with toluene, andat the end of a 24-hour collection period, centrifuged in

* Work supported by a grant from the Department ofMedical Care, National Foundation, and by grant no.2A-5154, N.I.A.M.D., U. S. Public Health Service;published in part in abstract form (1).

t Trainee in Arthritis, N.I.A.M.D., U. S. Public HealthService.

a washing machine and then extracted three times withmore water. The pooled extract and washings had afinal volume of about four liters. A suitable sample wasthen taken for determination of hydroxyproline. Re-coveries of this amino acid, carried out by soaking diaperswith a known amount of urine, averaged 97 per cent.

Hospitalized controls. Individuals with diseases inwhich marked alteration of collagen synthesis, break-down, or excretion might be present were not acceptedas controls. Accordingly, patients with anorexia, mal-nutrition, acute infection, renal disease, dystrophy, andinborn errors of connective tissue metabolism were ex-cluded. Urines from hospitalized infants were collectedby means of a metabolic bed or by use of a finger cot at-tached to polyethylene tubing.

In eleven hospitalized, adult control subjects, the break-down of diseases was as follows: myocardial infarction, 1;convalescent rheumatic fever, 1; inactive pulmonary tu-berculosis, 2; post-delirium tremens, 1; osteoarthritis, 2;compensated alcoholic cirrhosis, 1; hypertensive cardio-vascular disease, 1; arteriosclerotic heart disease, 1; andscleroderma, 1. In 44 hospitalized children, the follow-ing conditions were represented: club foot, 4; post-poliomyelitis under rehabilitation, 14; convalescent sal-monellosis, 1; convulsive disorder, 3; inactive rheumaticfever, 1; convalescent meningitis, 4; post-arsenic poison-ing, 1; convalescent pneumonia, 3; pes planus, 1; cysticfibrosis, 1; scoliosis, 1; croup, 1; inactive tuberculosis, 1;bronchitis, 3; rheumatoid arthritis, 1; infectious mononu-cleosis, 1; cellulitis, 1; psychiatric disorder, 1; and sickle-cell anemia, 1.

A gelatin-free diet was instituted at least one day be-fore the collection period. Urines were collected undertoluene and samples stored at - 150 C. Unless other-wise specified, values represent averages of two consecu-tive 24-hour collections.

Total and free hydroxyproline were determined by themethod of Prockop and Udenfriend (8) with the follow-ing modifications. To a 2-ml sample containing 2 to 10,ug of hydroxyproline, there were added 2 g of solid po-tassium chloride, 0.3 ml of a 10 per cent alanine solution,pH 8.7, and 0.7 ml of 0.25 N borate buffer, pH 8.7. Oxi-dation was carried out with 0.6 ml of 0.2 Mchloramine-Tin methyl Cellosolve for 20 minutes at room tempera-ture. The reaction was stopped by addition of 2 ml of3.6 M sodium thiosulfate. The samples were then ex-tracted once with 3.3 ml of toluene and the toluene layerdiscarded. A second volume of 3.3 ml of toluene was

1928

RELATIONSHIP BETWEENURINARY HYDROXYPROLINEAND GROWTH

then added and the mixture allowed to stand in a boil-ing water bath for 30 minutes. Color development was

carried out by adding 0.6 ml of para-dimethylamino-benzaldehyde reagent to 1.7 ml of the toluene layer.Samples were analyzed in duplicate. Unless otherwisespecified, the values given in the text represent totalhydroxyproline.

Total urinary amino acids were determined by themethod of Troll and Cannan (9).

Nitrogen and potassium balance. The pituitary dwarfS. B. was subjected to a 5-week balance study. The foodsupply was obtained in advance to assure uniformity.The diet was gelatin-free and afforded 700 kg-cal daily.Urine and feces were collected each day and frozen.Nitrogen analysis of food, urine, and feces was carriedout by the combined macro-Kjeldahl and Conway dif-fusion techniques (10). Potassium was determined on

the flame photometer with internal standards.

RESULTS

Normal growth. Table I gives the daily totalurinary hydroxyproline excretion observed inadult control subjects, both normal and hospital-ized (Figure 1), and Table II, the correspondingvalues for control children. In the latter, the 24-hour excretion increased with age, but decreasedagain in the adult age group, where the valueswere similar to those of 0- to 1-year-old children.In the 10- to 14-year group, in which the factor

140 0'4 120

\t0:> ' -vow* .¶71(6 v*A7 v

t 40 _ v v v

o 20 -

V.- .:oVChildren Children Children NornalI0-tyr (-lOyrs 10-14yrs AdduItr

Dwtr fJ

FIG. 1. DAILY HYDROXYPROLINEEXCRETION IN CHIL-

DREN, ADULTS, AND DWARFS. Horizontal lines representmean values, *, males, and V, females.

of size in relation to the adult group was least sig-nificant, the average excretion was three timesgreater than in the adults.

Whenexcretion was calculated per square meterof body surface to correct for differences in size,there was no overlap between adults and childrenin any age group, with results statistically sig-nificant at the 0.001 probability level for all age

groups. It is interesting to note that the highervalues obtained in children in the 0- to 1- and 10-to 14-year groups, when compared to the 1- to 10-

TABLE I

Daily urinary hydroxyproline in control adults

Hydroxyproline in mg Hydroxyproline in mg/M2Body surface

Group Number area Mean Range Mean Range

mneNormal

adults* 12 1.49-2.19 32.9 14.5-54.6 18.1 9.3-30.6Hospitalized

adults 11 34.7 19.3-59.1

* One 24-hour specimen was analyzed.TABLE II

Daily urinary hydroxyproline in control children

Hydroxyproline in mg Hydroxyproline in mg/M2Body surface

Group Number Age area Mean Range Mean Range

years m2Normal * 7 0-1 0.20-0.39 32.7 17.6- 43.0 102.2 48.0-130.0

21 1-10 0.44-1.30 49.0 14.9-150.0 66.1 41.9-145.0

Hospitalized 9 0-1 0.25-0.45 31.2 21.3- 52.3 95.1t 53.0-141.223 1-10 0.49-1.24 47.7 24.0-102.2 63.1 42.5- 85.012 10-14 0.99-1.64 103.7 67.5-169.0 80.5t 55.0-111.5

* One 24-hour urine specimen was analyzed.t p < 0.01 with respect to excretion in the 1- to 10-year-old group, calculated by t test.

1929

H. E. JASIN, C. W. FINK, W. WISE, AND M. ZIFF

TABLE III

Daily urinary hydroxyproline in dwarfism

Body surfaceDiagnosis Patient Age area Hydroxyproline

years m2 mg mg/m2Pituitary S.B. 8.3 0.55 14.1 25.6

dwarfism W.R. 10.5 0.86 41.3 48.0

Cretinism J.B. 4.3 0.53 5.5 10.4L.T. 0.3 0.29 5.6 19.6B.S. 5.3 0.70 11.8 16.9

Cockayne's C.E. 6.5 0.42 8.2 19.5syndrome (11)

Arthrogryposis G.L. 1.5 0.32 14.3 46.1B.L. 1.5 0.32 14.9 46.6

Intra-uterine runt J.Y. 3.0 0.38 14.1 37.1

Hurler's syndrome L.S. 3.8 0.45 17.3 38.4

year group, correspond to periods of more rapidgrowth.

Dwarfism. Table III lists the findings in tenchildren with dwarfism of various etiologies.Except for one of the pituitary dwarfs, W. R., allvalues are markedly decreased, both on an abso-lute basis and per square meter, when comparedwith those of children of the same age or size.The patient W. R. and the two patients with ar-

throgryposis, who were twins, were known to begrowing slowly. As shown in Figure 1, the de-creased hydroxyproline excretion values in thedwarf group approximated those of normal adults.

Effect of treatment of endocrine dwarfism on

excretion

Pituitary dwarfism. (Case 1)-S. B., an 8.3-year-old white girl, had developed very slowly.On admission, height was 89.5 cm, weight, 12.4kg, and bone age, 3.5 years. The patient was

small but well proportioned. Physical examina-tion was otherwise negative. Laboratory find-ings were: protein-bound iodine (PBI), 5.2 to 6.3jug per cent (normal, 4 to 8 jug per cent); 24-hourradioactive iodine uptake (RAI), 14.9 per cent(normal, 10 to 30 per cent); urinary 17-ketoster-oids, 1.1 mg per 24 hours (normal, 0.5 to 2 mg);17-ketogenic steroids, 1.0 mg per 24 hours (nor-mal, < 5 mg). After administration of 15 unitsof corticotrophin daily for 3 days, excretion of17-ketosteroids was 2.1 mg and of 17-ketogenicsteroids, 5.9 mg per 24 hours. The diagnosis was

pituitary dwarfism, presumed to be due to iso-lated growth hormone deficiency.

The patient was placed on a controlled, gelatin-free diet, and after 2 weeks of weight stabiliza-tion, 2.5 mg of human growth hormone 1 was ad-ministered intramuscularly 6 days a week, for 4weeks. At the end of treatment, the patient hadgained 700 g and grown 1.5 cm. Potassium andnitrogen balance studies indicated an immediateincrease in retention of these elements, more pro-nounced in the first week of treatment.

Figure 2 shows the levels of total urinary hy-droxyproline in the periods before, during, andfor three weeks after administration of growthhormone. Of note are the consistently low values,averaging 14.1 mg daily, observed during the con-trol period, and the prompt rise in excretion afterthe start of treatment. At the end of the treat-ment period, the patient had achieved a urinaryhydroxyproline level of 63.5 mg in 24 hours. Thecalculated value of 115.5 mg per m2 correspondsto a level attained by children only during pe-riods of rapid growth (see Table II). That thisincrease was not due to a nonspecific rise in uri-nary amino acids is shown by the fact that theseincreased only 50 per cent (see Figure 2), whilehydroxyproline rose more than fourfold. Threeweeks after growth hormone administration wasdiscontinued, hydroxyproline values dropped tocontrol levels. Free hydroxyproline of the urine

1 We thank the Endocrinology Study Section, N.I.H.,for generous supplies of this hormone.

1930

RELATIONSHIP BETWEENURINARY HYDROXYPROLINEAND GROWTH

6 12 18 24 30 36 42 41Day.s

FIG. 2. EFFECT OF GROWTHHORMONEON EXCRETIOI'HYDROXYPROLINEAND TOTAL AMINO ACIDS IN PITUIT

DWARFISM(CASE 1, S. B., ?, 8.3 YEARS). I0,resents hydroxyproline, and O---0, total urinary anacids.

remained unchanged during treatment, making1 to 3 per cent of the total.

(Case 2)-W. R., a 10.5-year-old whitegrew normally during the first year of life.ginning at 14 months of age, he had a numbethospital admissions because of convulsions asciated with hypoglycemia. At 3.7 years, tryment with 10 mg of hydrocortisone daily wasgun and continued intermittently until 4 webefore admission, when steroid treatment i

discontinued. On admission, height was 1Ccm and weight, 30.0 kg. Growth rate during3 years before admission had been half the rmal rate. At 7.5 years, RAI uptake was 28.1cent, and urinary 17-ketosteroids, 2.3 mg perhours. Laboratory findings on admission wEurinary 17-hydroxy-corticosteroid excretion,to 1.8 mg per 24 hours (normal, 2 to 8 mg); lnary 1 1-desoxy-corticosteroids (normal, 0 tFmg per 24 hours); and plasma 1 1-desoxy-cccosteroids (normal, 0 to 2.5 ug per cent), urtectable. After oral administration of 1 gmethopyrapone (SU-4885, Ciba) in 4 dividoses over a 24-hour period, urinary 17-hydrocorticosteroid excretion was 3.0 mg per 24 hoturinary 11-desoxy-corticosteroids, 0.9 to 1.5per 24 hours (normal, > 8 mg); and plasmadesoxy-corticosteroids, 1.0 jug. per cent (norr> 8 Mug per cent). After administration ofunits of corticotrophin gel daily for 3 days, 1

nary 17-hydroxy-corticosteroid excretion was

mg per 24 hours. The diagnosis was pituitaryt dwarfism with combined growth hormone and* adrenocorticotrophin deficiency.

After a 2-week control period on a gelatin-freediet, 2.5 mg of human growth hormone was ad-

> ministered intramuscularly, 6 days a week, for 4so weeks. At the end of this period, the patient hadt gained 900 g and grown 1.2 cm.

Daily excretion of hydroxyproline averaged41.3 mg before administration of growth hor-mone (Figure 3). The peak values reachedduring the treatment period were three timesgreater. Three weeks after the hormone was dis-continued, excretion had fallen to near that of the

qOF control period. The hydroxyproline values of'ARYpeid yrxyrlnrep- 100 to 155 mg per m2 attained during the treat-nino ment period corresponded to those of very ac-

tively growing children.Cretinism. (Case 3)-J. B., a 4.3-year-old

up white girl, grew normally until age 2, when growthappeared to stop. On admission, physical find-

boy ings were typical of severe hypothyroidism.Be- Height was 88.5 cm and weight, 11.7 kg. Labora-r of tory findings: PBI, 1.1 pug per cent; RAI uptake,so- 4.2 per cent; and RAI uptake after administrationeat- of 5 units of thyrotrophin daily for 3 days, 1.1 perbe- cent. The diagnosis was primary hypothyroidism.reks Nine weeks after the start of treatment withwas desiccated thyroid, symptoms had disappeared,)6 .8 and the patient had grown 4.0 cm.the From a very low value of 5.5 mg per 24 hours,ior- the excretion of hydroxyproline had risen 17-foldper after 10 weeks of treatment (Figure 4). The24 value of 180 mg per m2 is greater than the high--re:1.4

uri-o 2rti-

ide-of

Ided)xy-

ars;mg11-

mal,15

uri-7.4

FIG. 3. EFFECT OF GROWTHHORMONEON EXCRETIONOF HYDROXYPROLINEIN PITUITARY DWARFISM (CASE 2,W. R., o", 10.5 YEARS).

1931

H. E. JASIN, C. W. FINK, W. WISE, AND M. ZIFF

4 4

v120

N

.,m 80

60

4 0-

Weeks

FIG. 4. EFFECT OF DESICCATED T

OF HYDROXYPROLINE IN PRIMA

(CASE 3, J. B., °, 4.3 YEARS).

est observed in the entire child(see Table II).

(Case 4)-L. T., a 4-monthadmitted because of decreaseand peculiar facies. Physical echaracteristic findings of seve

PBI was 1.0 jug per cent. '

congenital cretinism. Desiccatministered in a daily dosage of30 mg for 2 weeks, 45 mg for 4

thereafter. After 7 weeks of trwas 65.0 cm and weight, 6.9 1

After 8 weeks of treatmen'droxyproline excretion had incto 47.5 mg. The latter value e:

meter was 167.0 mg, and iswhen compared with the cor

group.

(Case 5)-B. S., a 5.3-yearreceived desiccated thyroid fopothyroidism intermittently s

months before admission, tr(rupted. On admission, heirweight, 21.0 kg, and bone age

PBI was 1.7 Mug per cent andper cent. After treatment vwwas resumed, the symptoms i

TABLE IV

Daily urinary hydroxyproline in cdesiccated thyroid (L. T., 9

and 10 weeks later the patient had grown 2.25 cm.

i The diagnosis was cretinism.4t Urinary hydroxyproline excretion averaged

/ 11.8 mg per 24 hours before treatment. Thyroid

{, extract was administered in dosages from 15 in-, creasing to 150 mg daily. At the end of 8 weeks,a the excretion had risen to 71.7 mg, or 93.0 mg

60 per i2, a value characteristic of the excretion of8 10 i actively growing children.

HYROID ON EXCRETION Acromegaly. (Case 6)-T. B., a 43-year-old.RY HYPOTHYROIDISM white man, was admitted because of unremitting

headaches of 4 years' and muscle weakness of 3years' duration. During the preceding 9 years,

[hood control group he had noticed gradual enlargement of the nose

and fingers. Physical examination showed typi--old white girl, was cal acromegalic features. Pertinent laboratoryd physical activity laboratory findings were: FBS, 109 mg per cent;Examination showed serum phosphorus, 3.9 to 4.6 mg per cent; PBI,re hypothyroidism. 5.4 ,ug per cent; and RAI uptake, 15.1 per centThe diagnosis was in 24 hours. Glucose tolerance curve was of the:ed thyroid was ad- diabetic type. X-ray findings were characteristic15 mg for 2 weeks, of acromegaly.

2 weeks, and 60 mg The patient was treated with irradiation over thereatment, her length sella turcica, with a 3,200-roentgen tumor dosekg. over a 1-month period.t (Table IV), hy- The mean value of urinary hydroxyproline ob-reased from 5.6 mg served before irradiation, 107 mg per 24 hours,xpressed per square or 52.0 mg per m', is well above the highest level

markedly elevatedrresponding control

140_'-old white boy, hadr symptoms of hy-ince age 2. Eighteatment was inter-ght was 94.0 cm,

, 1.5 years. Serumcholesterol, 454 mg

rith thyroid extractimproved promptly,

cretinism treated withage 4 months)

Weeks oftreatment Hydroxyproline

mg mg/m2

0 5.6 19.61 15.6 55.08 47.5 167.0

lb 120

100

It 80Na

0

as6080

as 40

20

_

_

0

0

V TV 0°0~~~~~V0 00

0

* .

VV:

AduUCont rol y

AcromegOct ly

FIG. 5. URINARY HYDROXYPROLINEIN PATIENTS WITHACTIVE AND INACTIVE ACROMEGALYAND IN ADULT CONTROLSUBJECTS. Horizontal lines represent mean values; 0,

male with active acromegaly; 0, males with inactiveacromegaly; 0, control males; and V, control females.

I Ih /

1932

. - - _

_

RELATIONSHIP BETWEENURINARY HYDROXYPROLINEAND GROWTH

of excretion found in the group of normal adults(Figure 5). Three months after the course ofradiation therapy ended, the excretion had de-creased to 58.3 mg, or 28.3 mgper M2, and a simi-lar value of 60.8 mg, or 29.5 mg per M2, was ob-tained 6 months later. Both post-treatment valueswere in the upper range of normal. Free urinaryhydroxyproline in the pretreatment urines repre-sented 1 to 2 per cent of the total.

Five additional patients with the diagnosis ofacromegaly were studied in whom there was noevidence of clinical activity. One had an elevatedtotal excretion of 84.1 mg per 24 hours, and fourshowed excretion values in the upper range ofnormal (see Figure 5).

DISCUSSION

Growth in childhood expressed in increment ofbody surface area per year is especially rapid inthe first year of life and above age 10 for girls and12 for boys, and relatively slower in the inter-vening period (12).2 Accordingly, the excretiondata in children representing the combined valuesof both sexes were grouped in 0- to 1-, 1- to 10-and 10- to 14-year age groups. Significant dif-ferences in absolute excretion between childrenand adults were noted only in the 10- to 14-yeargroup, where size difference is less marked.When excretion was calculated per unit surfacearea, however, to correct for the influence of size,significant elevation above the adult average wasnoted in all childhood age groups. Surface areawas chosen as a parameter of size because it wasthought to be a more reliable index of connectivetissue mass than body weight.

The excretion values per square meter given inTable II are significantly greater in the 0- to 1- and10- to 14-year groups than in the 1- to 10-yeargroup, indicating a correlation between hydroxy-proline excretion per unit surface area and growthrate, since, as mentioned above, growth is knownto be more rapid in these two age periods. Thesefindings suggest that when sufficient data areavailable, it will be possible to relate excretionper unit surface area to the normal growth curve.

All ten children with dwarfism showed low ex-

2 Values for the fiftieth percentile of height and weightin normal children (12) were used to calculate surfacearea.

cretion of hydroxyproline. On an absolute basis,the values in nine of these fell between 5.5 and17.3 mg, or below the range of the control valuesfor normal children. Seven of the ten had lowvalues per unit surface area, and the remainingthree, who were known to be growing slowly,had borderline values. The low excretion ob-served in non-growing or slowly growing childrenis further evidence for a relationship betweenhydroxyproline excretion and growth.

Most impressive was the rise in excretion ofhydroxyproline following hormonal treatment ofnon-growing endocrine dwarfs. When growthhormone was administered to two pituitarydwarfs and desiccated thyroid to three cretins, ex-cretion of hydroxyproline rose dramatically in allcases, and this was accompanied by objective evi-dence of growth. All five dwarfs attained levels ofexcretion that were characteristic of actively grow-ing children. In the case of two of the cretins,the values calculated per square meter were thehighest ever noted by this laboratory in any agegroup. When, in the case of the two pituitarydwarfs, administration of growth hormone washalted, excretion returned to pretreatment valueswithin 3 weeks.

Further evidence of a relationship between hy-droxyproline excretion and growth was seen inthe markedly elevated values observed in the pa-tient with active acromegaly. Before treatment,these were three times the adult average; 3 monthsafter irradiation, they had fallen to near normallevels. The data in five patients with clinicallyinactive acromegaly were more difficult to inter-pret. Excretion ranged between 49.7 and 84.1mg, that is, from above normal to the upper lim-its of normal. Whether this indicates residual ac-tivity, undetectable by the usual criteria, is notclear.

Any mechanism attempting to explain the ob-served relationship between growth rate and ex-cretion of hydroxyproline ought to take into con-sideration the presence of increased amounts ofsoluble collagen in the tissues of growing animals(13-16), presumably as a consequence of an in-creased rate of synthesis of this protein. Thisfraction has a high turnover rate and is the pre-cursor of fibrous collagen. It is also known thaturinary hydroxyproline is increased in young ani-mals as compared with adults (5, 6). These con-

1933

H. E. JASIN, C. W. FINK,

siderations, as well as the results reported in thispaper, agree with an earlier suggestion (2) thatthe increased excretion associated with growthrepresents peptides derived from soluble collagen.

Data recently obtained in this laboratory inrats with osteolathyrism (7) also support thepoint of view above. These animals had in-creased amounts of neutral salt-extractable col-lagen in the skin and excreted urinary hydroxy-proline at elevated levels without gross differ-ences in growth rate from control animals. Thisfinding indicates that hydroxyproline peptide ex-cretion rises with an increase in soluble collageneven when the latter change occurs as a result offactors other than growth. In this connection, anincreased excretion of hydroxyproline has re-cently been reported in patients with Paget's dis-ease and hyperparathyroidism (17), conditionsin which the rate of formation of new collagen inbone is presumably increased.

Although the arguments presented stronglysuggest that the increased excretion during growthdepends on an increase in soluble collagen, it isdifficult to evaluate the contribution of the fibrouscollagen to urinary hydroxyproline. Although thisfraction turns over very slowly (18), it is presentin large amounts. Lindstedt and Prockop (5)have recently demonstrated a urinary hydroxy-proline fraction in adult rats with a turnover rateof about 300 days, which they suggested was de-rived from the fibrous collagen pool. It is pos-sible, therefore, that fibrous collagen does con-tribute to urinary hydroxyproline, and since thereis little soluble collagen in adult tissue (19), thecontribution from mature fiber would be propor-tionately greater in this group than in children.

In considering other possible explanations forthe increased excretion of hyroxyproline duringgrowth, one cannot rule out the possibility thatrenal factors may play a part in the phenomenadescribed, since it is known that growth hormone(20, 21) and thyroid hormone (20, 22) may in-fluence renal function. Against this possibility,however, is the well documented evidence thatrenal clearance and glomerular filtration rate ex-pressed per unit surface area, in contrast to hy-droxyproline excretion, are low in the first yearof life and shortly thereafter attain adult levelsthat remain constant from then on (20, 23).Also, it may be pointed out, total amino acid ex-

cretion, mainly in the form of peptides (24), rosemuch less than hydroxyproline excretion whenmeasured in one of the pituitary dwarfs. Finally,the increased excretion noted in osteolathyrism isevidence against a renal factor, since hormonalinfluences capable of affecting renal function werepresumably not involved in the lathyritic process.

SUMMARY

Total hydroxyproline excretion has been deter-mined in normal and hospitalized adults and chil-_dren, in children with dwarfism, and in patientswith acromegaly.

Total excretion was significantly greater inchildren in the 10- to 14-year group than in adults.When calculated per unit body surface area, itwas greater in all childhood age groups. Peaklevels of excretion per square meter coincidedwith periods of active growth.

Children with dwarfism showed decreased ex-cretion of hydroxyproline. Administration ofgrowth hormone to two children with pituitarydwarfism and of thyroid hormone to three chil-dren with cretinism resulted in a prompt rise inexcretion, which attained levels characteristic ofrapidly growing children. After cessation ofgrowth hormone administration, excretion fell topretreatment levels.

One patient with active acromegaly excretedmarkedly increased amounts of hydroxyproline.After irradiation therapy, these fell toward nor-mal. Five patients with clinically inactive acro-megaly showed levels elevated in one case andotherwise in the upper range of normal.

We conclude that there is a close relationshipbetween rate of growth and urinary hydroxypro-line excretion, and suggest that the increased ex-cretion is a result of the presence during growthof an increased amount of metabolically active,soluble collagen in the tissues, which is availablefor breakdown. The results indicate that meas-urement of urinary hydroxyproline may providea useful index of growth activity.

ACKNOWLEDGMENTS

We wish to thank Dr. J. Donald Smiley for his in-valuable help and advice, the staff of the Texas ScottishRite Hospital for their cooperation, and Drs. JamesHyslop and Norman Kaplan for their kind assistance.

1934 W. WISE, AND M. ZIFF

RELATIONSHIP BETWEENURINARY HYDROXYPROLINEAND GROWTH

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2. Ziff, M., Kibrick, A., Dresner, E., and Gribetz, H. J.Excretion of hydroxyproline in patients with rheu-matic and non-rheumatic diseases. J. clin. Invest.1956, 35, 579.

3. Neuman, R. E., and Logan, M. A. The determinationof collagen and elastin in tissues. J. biol. Chem.1950, 186, 549.

4. Prockop, D. J., and Sjoerdsma, A. Significance ofurinary hydroxyproline in man. J. clin. Invest.1961, 40, 843.

5. Lindstedt, S., and Prockop, D. J. Isotopic studieson urinary hydroxyproline as evidence for rapidlycatabolized forms of collagen in the young rat.J. biol. Chem. 1961, 236, 1399.

6. Martin, G. R., Mergenhagen, S. E., and Prockop,D. J. Influence of scurvy and lathyrism (odora-tism) on hydroxyproline excretion. Nature 1961,191, 1008.

7. Jasin, H. E., and Ziff, M. Relationship betwen solu-ble collagen and urinary hydroxyproline in therat. Proc. Soc. exp. Biol. In press.

8. Prockop, D. J., and Udenfriend, S. A specificmethod for the analysis of hydroxyproline in tis-sues and urine. Analyt. Biochem. 1960, 1, 228.

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11. McDonald, W. B., Fitch, K. D., and Lewis, I. C.Cockayne's syndrome. An heredo-familial dis-order of growth and development. Pediatrics 1960,25, 997.

12. Nelson, W. E. Textbook of Pediatrics, 7th ed.Philadelphia, W. B. Saunders, 1959, p. 13.

13. Gross, J. Studies on formation of collagen. II. Theinfluence of growth rate on neutral salt extractsof guinea pig dermis. J. exp. Med. 1958, 107, 265.

14. Banfield, W. Effect of growth hormone on aceticacid-extractable collagen of hamster skin. Proc.Soc. Exp. Biol. (N. Y.) 1958, 97, 309.

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